Flexibility in mua transposase family protein structures: Functional mapping with scanning mutagenesis and sequence alignment of protein homologues

Tiina S. Rasila, Mauno Vihinen, Lars Paulin, Saija Haapa-Paananen, Harri Savilahti (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

3 Citations (Scopus)

Abstract

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.
Original languageEnglish
Article numbere37922
JournalPLoS ONE
Volume7
Issue number5
DOIs
Publication statusPublished - 2012
MoE publication typeA1 Journal article-refereed

Fingerprint

Transposases
Mutagenesis
Sequence Alignment
sequence alignment
protein structure
mutagenesis
Scanning
DNA
Integrases
Proteins
proteins
Genomics
Molecular Biology
genomics
DNA Cleavage
DNA Transposable Elements
transposition (genetics)
structure-activity relationships
Biotechnology
Molecular biology

Cite this

Rasila, Tiina S. ; Vihinen, Mauno ; Paulin, Lars ; Haapa-Paananen, Saija ; Savilahti, Harri. / Flexibility in mua transposase family protein structures : Functional mapping with scanning mutagenesis and sequence alignment of protein homologues. In: PLoS ONE. 2012 ; Vol. 7, No. 5.
@article{c1d010ba786d4ea8aac224cd3e9d1bc0,
title = "Flexibility in mua transposase family protein structures: Functional mapping with scanning mutagenesis and sequence alignment of protein homologues",
abstract = "MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.",
author = "Rasila, {Tiina S.} and Mauno Vihinen and Lars Paulin and Saija Haapa-Paananen and Harri Savilahti",
year = "2012",
doi = "10.1371/journal.pone.0037922",
language = "English",
volume = "7",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "5",

}

Flexibility in mua transposase family protein structures : Functional mapping with scanning mutagenesis and sequence alignment of protein homologues. / Rasila, Tiina S.; Vihinen, Mauno; Paulin, Lars; Haapa-Paananen, Saija; Savilahti, Harri (Corresponding Author).

In: PLoS ONE, Vol. 7, No. 5, e37922, 2012.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Flexibility in mua transposase family protein structures

T2 - Functional mapping with scanning mutagenesis and sequence alignment of protein homologues

AU - Rasila, Tiina S.

AU - Vihinen, Mauno

AU - Paulin, Lars

AU - Haapa-Paananen, Saija

AU - Savilahti, Harri

PY - 2012

Y1 - 2012

N2 - MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

AB - MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

U2 - 10.1371/journal.pone.0037922

DO - 10.1371/journal.pone.0037922

M3 - Article

VL - 7

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 5

M1 - e37922

ER -